The long-term objective of our research is to understand the biological function of a family of transaltional modulators known as PABP interacting protiens (Paips) which were first cloned in our laboratory. These proteins mediate their effects on translation by interacting with the poly(A) binding protein (PABP). Previous work has shown that Paipi stimulates translation, whereas Paip2A and Paip2B inhibit translation. The specific aims of this proposal are to further our understanding of the molecular mechanism of Paip action, to discover how Paip function is regulated, and to elucidate the biological significance of this family of proteins. A variety of in vitro and in vivo experiments will be carried out to study the Paip1PABP interactions, to dissect their mechanism of action, and to identify novel Paip interacting proteins. Paip expression and function will be inhibited by the RNA interference technique and the use of small cell-permeable peptides. Paip phosphorylation will be explored to elucidate the signaling pathways impinging upon Paip function. To this end, phosphopeptide mapping and 2-dimensional isoelectric focusing and SDS-PAGE will be used to study the phosphorylation states of the Paips under various environmental conditions and upon treatment with pharmacological kinase inhibitors. The phosphoresidues in the proteins will be identified and mutated. Resulting proteins will be assayed using in vitro and in vivo translation experiments to discover their functional importance. Elucidation of the physiological role of the Paips will be pursued by Paip overexpression and knockout (KO) experiments in Drosophila. Paip null flies will be generated by P-element insertion or homologous recombination. The generation of KO mice devoid of the individual Paip genes will also be pursued by homologous recombination. In the case of Paip2A and Paip2B, which are functional homologs, a Paip2AIPaip2B KO mouse will be generated. KO mice will be analyzed for phenotypic abnormalities.